PROJECT SUMMARY/ABSTRACT The pruning of dendritic spines, and the glutamate synapses they receive, on prefrontal cortex (PFC) layer 3 pyramidal neurons (L3PNs) during adolescence is thought to play a key role in the progression of cognitive dysfunction and the onset of psychosis in schizophrenia. This idea is based, in part, on findings that the densities of L3PN spines and layer 3 axospinous synapses in the primate (but not rodent) PFC decline by ~40% between late childhood and early adulthood, coinciding with the maturation of PFC-mediated cognitive abilities that are impaired in schizophrenia. In monkeys, L3PNs comprise distinct subtypes defined by the cortical projection target of their principal axon. However, prior studies of synaptic pruning in primate PFC did not differentiate between L3PN subtypes, which likely differ in developmental trajectories of synaptic maturation. For example, L3PNs projecting to the ipsilateral parietal cortex (pL3PNs) contribute to the PFC- parietal network mediating cognitive functions that improve substantially across adolescence, when PFC- parietal functional connectivity is enhanced. Thus, synaptic pruning in pL3PNs during adolescence could be critical for the maturation of the PFC-parietal network. In contrast, callosal projection PFC L3PNs (cL3PNs) form the inter-hemispheric network that is refined early and achieves mature features before adolescence. Thus the goal of the proposed studies is to determine the cell type-specificity, functional consequences and molecular correlates of synaptic pruning in the primate PFC during adolescence. In each study, pL3PNs and cL3PNs in monkey PFC are retrogradely-labeled with fluorescent microspheres in vivo, and subjected to in vitro electrophysiology, biocytin filling and confocal imaging or captured by laser microdissection for RNAseq analysis. Using this innovative approach, the following four hypotheses (H) will be tested: H1) Spines on pL3PNs receiving cortical inputs are preferentially pruned during adolescence. In contrast, spines on cL3PNs are not pruned during adolescence. H2) Pruning during adolescence is associated with increases in spine volume and excitatory synaptic current amplitude selectively on pL3PNs. H3) Strengthening of cortical synaptic inputs during adolescence enhances behaviorally-relevant activity of pL3PNs. H4) The transcriptional profiles of pL3PNs and cL3PNs follow distinct developmental trajectories that reflect differences in spine pruning and result in cell type-specific gene expression patterns in the adult PFC that can be used to identify L3PN subtypes in human PFC. Given recent evidence that the genetic liability for schizophrenia alters pruning, the proposed studies will provide critical information to guide the conduct and interpretation of separately-funded studies examining the cell type-specificity and developmental origin of spine deficits in schizophrenia.